Part:BBa_K3772033
TKL2-VS-SAG1t expression cassette
Express valencene synthase under the control of promoter TKL2 (BBa_K3772023). We will use different promoters to initiate transcription of valencene synthase gene. Target promoters were screened by yield analysis of valencene. Then, target promoters will be changed the upstream regulatory sequence to achieve the maximum yield of valencene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 273
Illegal AgeI site found at 1805 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This Composite Part is composed of basic parts CDC19 (BBa_K3772015), valencene synthase coding sequence (BBa_K3772005) and terminator SAG1t (BBa_K3772014). ) and the terminator SAG1t (BBa_K3772014).14 yeast promoters with potential in different metabolic pathways (glucose degradation, gluconeogenesis and pentose phosphate pathways) were found through literature review [1]. The Promoter-VS-SAG1t expression cassette was used to allow the transcriptional expression of valencene synthases in the presence of their respective promoters. Since valencene synthase is a key enzyme in the rate limiting reaction of naringenone synthesis, we expect that increased expression of valencene synthase will more likely lead to a smoother transformation of our project. Therefore, we demonstrated the difference in transcriptional intensity of valencene synthase coding sequence by verifying the yield of valencene for these 14 promoters. The promoters with higher intensity will be analyzed more closely and will be the subject of our next transformation or auxiliary depending on their properties.
Because of the instability of regulatory element substitution across subspecies, the promoters in the Promoter-VS-SAG1t expression cassette are derived from Saccharomyces cerevisiae itself. Different types of carbon sources have different activation effects on cells, making them express different transcription activators. Certain transcription activators bind to UASs of promoters, recruit RNA polymerase, and regulate genes globally.
Because of the instability of substitution of regulatory elements across subspecies, the promoters in the Promoter-VS-SAG1t expression cassette are all derived from brewer's yeast itself. These promoters possess different characteristics due to their different carbon source responsive properties. Different types of carbon sources have different activation effects on cells, making them express different transcription Activators. Certain transcription Activators bind to UASs of promoters, recruit RNA polymerase, and regulate genes globally.
Fig.1:Search for natural promoters.
Characterization
Expression intensity of promoters
The product valenece was tested after incubation for 64 h in a 10 ml YPD system at 30 ℃ and 220 rpm inoculated with 0.05 OD of the engineered S. cerevisiae BJ5464-N strain. The results are shown in Fig.2.
Fig.2 Fermentation results-genome level LEU2::P-VS-T. In which it was observed that although the individual Promoter-VS-SAG1t had little effect on the growth of the strain, the intensity of the promoters varied widely. If we set the intensity of PCD1 was 100%, the intensity distribution of different promoters ranged from 3% to 74%. Among them, we selected PDC1p from Glycolysis pathway Promoters, ALD4p from Ethanol metabolism Promoters, SED1Lp from Other pathway Promoters and ZWF1 from Pentose Phosphate. These four Promoter-VS-SAG1t expression cassettes were subsequently analyzed for time curve and the ability to respond to different carbon sources.
Reference
[1] Nambu-Nishida, Y., et al., Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation. Journal of Bioscience and Bioengineering, 2018. 125(1): p. 76-86.
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